Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot. Generally, the blood components which participate in what has been referred to as the coagulation "cascade" are no proenzymes or zymogens, enzymatically inactive proteins, which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor. Coagulation factors that have undergone such a conversion and generally referred to as "active factors", and are designated by the addition of the letter "a" to the name of the coagulation factor (e.g. FVIIa).
Activated factor X (FXa) is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot. There are two systems, or pathways that promote the activation of factor X. The "intrinsic pathway" refers to those reactions that lead to thrombin formation through utilisation of factors present only in plasma. A series of protease-mediated activations ultimately generates factor IXa, which, in conjunction with factor VIIIa, cleaves factor X into Xa. FVIIa and its cofactor TF in the "extrinsic pathway" of blood coagulation effect an identical proteolysis. TF is a membrane bound protein and does not normally circulate in plasma. Upon vessel disruption, however, it is exposed and forms a complex with FVIIa to catalyse factor X activation or factor IX activation in the presence of Ca.sup.2+ and phospholipid (Nemerson and Gentry, Biochemistry 25:4020-4033 (1986)). While the relative importance of the two coagulation pathways in hemostasis is unclear, in recent years FVIIa and TF have been found to play a pivotal role in the initiation and regulation of blood coagulation.
FVII is a trace plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is catalytically inactive (Williams et al., J. Biol. Chem. 264:7536-7543 (1989); Rao et al., Proc. Natl. Acad. Sci. USA. 85:6687-6691 (1988)). Single-chain FVII may be converted to two-chain FVIIa by factor Xa, factor XIIa, factor IXa, FVIIa or thrombin in vitro. Factor Xa is believed to be the major physiological activator of FVII. Like several other plasma proteins involved in haemostasis, FVII is dependent on vitamin K for its activity, which is required for the gamma-carboxylation of multiple glutamic acid residues that are clustered in the amino terminus of the protein. These gamma-carboxylated glutamic acids are required for the metal-associated interaction of FVII with phospholipids.
The conversion of zymogen FVII into the activated two-chain molecule occurs by cleavage of an internal Arg152-IIe 153 peptide bond (Hagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416 (1986); Thim et al., Biochemistry 27:7785-7793 (1988)). In the presence of TF, phospholipids and calcium ions, the two-chain FVIIa rapidly activates factor X or factor IX by limited proteolysis.
It is often desirable to selectively block or inhibit the coagulation cascade in a patient. Anticoagulants such as heparin, coumarin, derivatives of coumarin, indandione derivatives, thrombin inhibitors, factor Xa inhibitors, modified FVII or other agents have previously been used.
Inhibition of coagulation is beneficial in a number of diseased states, for example during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), atherosclerosis and a host of other medical disorders. For example, heparin treatment or extracorporeal treatment with citrate ions (U.S. Pat. No. 4,500,309) may be used in dialysis to prevent coagulation during the course of treatment. Heparin is also used in preventing deep vein thrombosis in patients undergoing surgery. Treatment with heparin and other anticoagulants may, however, have undesirable side effects. Available anticoagulants generally act throughout the body, rather than acting specifically at the site of injury, i.e. the site at which the coagulation cascade is active. Heparin, for example, may cause severe bleedings. Furthermore, with a half-life of approximately 80 minutes, heparin is rapidly cleared from the blood, necessitating frequent administrating.
Other known anticoagulants comprise thrombin and factor Xa inhibitors derived from bloodsucking organisms. Antithrombins, hirudin, hirulog and hirugen are recombinant proteins or peptides derived from the leach Hirudo medicinalis, whereas the factor Xa inhibitor antistatin and the recombinant derivative rTAP are tick-derived proteins. Inhibitors of platelet aggregation such as monoclonal antibodies or synthetic peptides, which interfere with the platelet receptor GPIIb/IIIa are also effective as anticoagulants.
Bleeding complications are observed as an undesired major disadvantage of anti-thrombin, anti-factor Xa, as well as anti-platelet reagents. This side effect is strongly decreased or absent with inhibitors of the FVIIa/TF activity. Such anticoagulants comprise the physiological inhibitor TFPI (tissue factor pathway inhibitor) and modified FVII (FVIIai), which is FVIIa modified in such a way that it is catalytically inactive but still binds to TF and competes with active FVIIa.
In addition to the anticoagulants briefly described above, several naturally occurring proteins have been found to have anticoagulant activity. For example, Reutelingsperger (U.S. Pat. No. 4,736,018) isolated anticoagulant proteins from bovine aorta and human umbilical vein arteries. Maki et al. (U.S. Pat. No. 4,732,891) discloses human placenta-derived anticoagulant proteins.
The anticoagulant FVIIai has also been shown to have effect in suppressing or preventing restenosis. Proliferation of smooth muscle cells (SMCs) in the vessel wall is an important event in the formation of vascular lesions in atherosclerosis, after vascular reconstruction or in response to other vascular injury. For example, treatment of atherosclerosis frequently includes the clearing of blocked vessels by angioplasty, endarterectomy or reduction atherectomy, or by bypass grafting. These are surgical procedures in which atherosclerotic plaques are compressed or removed through catheterization (angioplasty), stripped away from the arterial wall through an incision (endarterectomy) or bypassed with natural or synthetic grafts. These procedures remove the vascular endothelium, disturb the underlying intimal layer, and result in the death of medial SMCs. Medial SMC proliferation and migration follow this injury into the intima, which typically occurs within the first few weeks and up to six months after injury and stops when the overlying endothelial cell layer is re-established. In humans, these lesions are composed of about 20% cells and 80% extracellular matrix. In about 30% or more of patients treated by angioplasty, endarterectomy or bypass grafts, thrombosis and/or SMC proliferation in the intima causes re-occlusion of the vessel and consequent failure of the reconstructive surgery. This closure of the vessel subsequent to surgery is known as restenosis.
For long term prophylactic treatment and increased compliance it is desirable to have access to low-molecular-weight compounds which may be administered via a route other than intravenously and which have an inhibitory effect on FVIIa-TF activity similar to that of FVIIai.
Thus, there is still a need in the art for improved compositions having anticoagulant activity which can be administered orally or otherwise non-intravenously at relatively low doses and which does not produce any undesirable side effects. The present invention fulfils this need by providing anticoagulants that act specifically on FVIIa/TF/FX complex formed at sites of injury.
It is also desirable to have access to a method for identifying compounds having an inhibitory action on FVIIa/TF activity.